The detection and quantitative determination of hydrogen peroxide and compounds which yield hydrogen peroxide as a result of chemical or enzymatic reactions are of importance in many areas. For example, they are important in the determination of hydrogen peroxide produced in the enzymatic assay of various chemical or biological substances (herein identified as analytes), such as glucose, cholesterol, uric acid, triglycerides, creatine kinase, creatinine, etc., in the presence of oxygen. The quantity of analyte present in a sample is determinable from the amount of hydrogen peroxide produced.
Known compositions and methods for determining hydrogen peroxide generally comprise a substance having peroxidative activity, e.g. peroxidase, and a color dye former which undergoes a detectable change (e.g. a color change) in the presence of hydrogen peroxide and peroxidase. Various materials which are known to undergo a detectable change in such conditions include monoamines, diamines, phenols and leuco dyes. Other hydrogen peroxide indicators include hydrogen donors (identified herein as color forming compounds) which react with color couplers to produce dyes.
U.S. Pat. Nos. 4,089,747 (issued May 16, 1978 to Bruschi) and 4,119,405 (issued Oct. 10, 1978 to Lam) relate to assays for hydrogen peroxide or analytes which generate hydrogen peroxide using a combination of a hydrazone and a color coupler. Use of the hydrazones described in these references in assaying whole blood has a disadvantage. Using the hydrazones, it is difficult to obtain detectable dyes which absorb electromagnetic radiation at relatively long wavelengths, i.e. greater than about 600 nm. Dyes formed with hydrazones generally absorb at shorter wavelengths and their detection is often hindered by various spectral interferents which are present in whole blood samples. The presence of these interferents would diminish the accuracy of the assay using hydrazones in testing whole blood or serum.